Simmental. The presence of antisperm Ig G and Ig A antibodies was

Transcription

1 499 DOI: /AVB L UDK: : / : THE INFLUENCE OF ANTISPERM Ig G AND Ig A ANTIBODIES FROM COWS SERA AND CERVICAL MUCUS ON BULL SPERM MOTILITY ***** Energetic Panacea Veterinary Clinic, Dubai, UE (Received 4 th April 2013) Ig G and Ig A antibodies (ASA) from the sera and cervical mucus of cows on bulls sperm motility. A total of 64 cows was included in the study and samples of sera and cervical mucus were collected on the day of Simmental. The presence of antisperm Ig G and Ig A antibodies was these results, cows were divided in groups as follows: cows with high or low ASA titer in their sera and cows with high or low ASA titer in the was further estimated by Computer Assisted Semen Analysis (CASA). antisperm antibodies depending on their origin and titer. Key words: antisperm antibodies, computer assisted sperm analysis, cows, subfertility INTRODUCTION tract disease. A repeat-breeder (RB) is a cow which shows a reduced probability of conception while all other factors are optimal (Casida, 1961) and thus require more attempts of AI to achieve pregnancy. If mated naturally, RB cows or heifers very et al., 1982). The possible causes of this phenomenon are numerous and include delicate nutritional misbalance, subtle hormonal disturbances and possibly immunological mechanisms. It was

2 500 Acta Veterinaria (Beograd), Vol. 63, No. 5-6, , postulated that enhanced immunological reactivity to sperm or semen extender antigens might be one of the reasons for RB (Park and Hunter, 1977). In natural mating the immune response to sperm antigens usually does not occur due to the low sperm and seminal plasma constituents immunogenicity (Landers et al., 1994). In AI chances for immunization of females to sperm antigens are enhanced because seminal plasma is diluted several times, thus et al., 1992) and new antigens originating from semen extenders are added. We et al., spermatozoa differ in antigenicity if different semen extenders are used for semen preparation in AI technology. An understanding of the functions of the mucosal immune system in the female reproductive tract is essential for dealing with problems of infertility and sub-fertility. One of the major problems, in both human and animal reproduction, is sterility with no obvious symptoms (unexplained infertility) that can be caused by anti-sperm antibodies (ASA) in seminal plasma, cervical mucus, uterine et al., 1992). The role of ASA in infertility and sub-fertility in mammalian species is still not clearly understood but it is well known that auto-immunization of the male and iso-immunization of the females Kim et al., 1999). Interference of ASA with reproductive processes may occur by impairment of sperm migration through the cervix, uterus and oviduct and by blocking adherence of spermatozoa to the surface of the zona pellucida of the oocyte as postulated by Schumacher (1998). The same author stated that genital tract secretions and that mucosal immunity may be operational mainly in the cervical compartment of the genital tract. Kim et al. (1999) demonstrated fertilization in vitro of anti-sperm antibodies raised by intensive immunization of bulls on semen quality. This phenomenon was described for numerous laboratory animals as reviewed by Hogarth (1982). Bratanov et al. (1975) proved that sera containing anti-sperm antibodies from infertile cows and women with unexplained infertility inhibit acrosomal proteolitic activity in vitro and therefore possibly may affect fertilization. Maas et al., (1989) demonstrated a close correlation between a positive sperm antibody test and poor post-coital test results in infertile couples. These authors concluded that determination of ASA in the CM must be regarded as an improvement in the diagnostic procedure in human infertility. We have

3 501 There is a strong evidence that in humans, sperm-mucus interactions can be affected by local ASA, especially of the Ig A class, both under in vitro and in vivo patients was not established, while those in seminal plasma or CM impaired the ability of sperm cells to penetrate CM (Eggert-Kruse et al., 1993). Check et al. (1991) demonstrated that the antifertility effect of ASA may be mainly the immobilization of sperm in the cervical mucus and thus intrauterine insemination may effectively correct the problem. The role of antisperm Ig A antibodies is also documented in the investigations conducted by Clarke (1988) and Kremer and Jager (1992). antispermatozoal antibodies in CM on fertility rates are very few. The elevation of inseminated cows according to the number of attempts was clearly demonstrated structure of sperm cells is changed due to the addition of different extenders, freezing and thawing procedures and reduction of seminal plasma volume. In Computer Assisted Semen Analisis (CASA) is a relatively new method that enables a precise measurement of the sperm motility and other sperm characteristics both in human or animal semen samples. It has been also used to et al., various extenders or extender supplements on the post-thaw motility, acrosome morphology and plasma membrane integrity by CASA (Hu et al., 2009). Recently method is very valuable in determining initial semen quality as well as changes during storage under different conditions (Tardif et al., 1997). By accumulating data by CASA it is now possible to investigate more accurately the relationship between sperm characteristics and fertility of males (Farrell et al., 1997). In this study we investigated the presence of ASA in the sera and cervical bull semen. MATERIAL AND METHODS Sera sampling: Sera were collected from Simmental or Busha cross breed temperature and centrifugation at 3000 rpm for 20 min. All samples wer kept frozen at -20 o C pending analisis. The animals were divided in two groups

4 502 Acta Veterinaria (Beograd), Vol. 63, No. 5-6, , according to the average value of ASA in sera or cervical mucus. All cows were previously inseminated with bull semen prepared for AI with egg-yolk extender. Cervical mucus sampling: Prior to AI, CM samples were collected by placing a sterile sponge swab in the near vicinity of the external cervix end by sterile et al., 2003). All samples were kept frozen at -20 o C until use. Semen sampling: Semen samples were collected from four Simmental bulls The semen had normal characteristics of motility, morphology and concentration and underwent the standard procedure of preparation for AI. Ejaculates were diluted with TRIS egg yolk extender at an average ratio of 1:10 and kept frozen at -196 o C before use. The IIF assay was performed according to Noel et al. (1974). This procedure was described in details earlier et al., 2003). Basically, after thawing the 12 (3 from each of 4 bulls) medium French straws for AI (0.25 ml), sperm cells were washed twice by centrifugation procedure in pre wormed PBS (ph 7.2, 37 o C), resuspended by Vortex and used for smear preparations. On the dried sperm cell smears, 10 L of sera or CM sample (inactivated at 56 o C for 20 minutes) was placed and incubated for 20 min at 37 o C in a wet chamber. Following incubation, the slides were washed three times (5 min) in PBS and dried at room temperature. In the second step, 10 L Ig A, ICN, USA, Cat No ) was placed on the slide and incubated again under the same conditions. Conjugation of anti Ig A antibodies with FITC (ICN, USA, Cat No F 4274) was performed according to The and Feltkamp (1970). After incubation, followed by the same washing procedure, the slides were kept in a dark and wet chamber until examined. As a positive control we used sera obtained by immunization of calves with content of straws prepared with TRIS egg yolk extender as described in detail elsewhere (Lazarevic et al., 2000). Calf sera obtained before immunization served as the negative control. Microscopic of the sperm cell was considered as a positive result and the last dilution giving a positive reaction was taken into account. Titer values were expressed according to Sjurin et al., (1984) as log 2 n (1:2 = 1, 1:4 = 2 etc). Sperm motility asessment Basic parameters of the bulls sperm motility were determined by CASA method (Computer Assisted Semen Analyses). Analyses were performed in series o C. We measured total sperm motility, progressive motility and velocity, tail movement amplitude and frequency by use of sperm-analyzer Hamilton - Thorn (HTM IVOS Version 2, No M 9368, USA)

5 503 negative control samples (inactivated serum of 3 months old calf). Results were calculated as a percent of the control values for a given group. Values obtained in two groups depending on the ASA titer (high or low titer) despite of the antibody class (Ig G or Ig A). Sera samples with higher total titer value of ASA (Ig A and Ig G together) then 1:16 and cervical mucus samples with titer above 1:64 were considered as high titer samples. Statistical analyses were performed after calculating mean values and was estimated by Student s t test. RESULTS The appearance of the control sample picture on the Hamilton-Thorn CASA analyzer is presented in Figure 1. Curved lines represent cells expressing progressive motility that was not altered by the presence of the control serum. Figure 1. Control sample on CASA analyzer (curved lines represent cells expressing progressive motility)

6 504 Acta Veterinaria (Beograd), Vol. 63, No. 5-6, , Figure 2. represents picture acquired from a cervical mucus sample containing a high titer of ASA. Only few sperm cells expressed progressive motility (curved lines). Figure 2. Cervical mucus sample with a high titer of ASA on CASA analyzer (curved lines represent cells expressing progressive motility) As can be concluded from the data presented below (Table 1) total sperm following incubation with sera containing high titers of ASA. in the cow s blood sera n = 11 n = 15 Blood sera n = 10 Thawed semen n = 13 X Progressive sperm motility was also lower in the native semen samples incubated with sera samples containing the high ASA titer (Table 2). These

7 505 when thawed semen was used for the test. Moreover, thawed semen had much lower progressive motility than native despite the ASA titer. ASA titer in the cow s blood sera Blood sera Thawed semen n = 11 n = 15 n = 10 n = 13 X ± ± ± ±15.08 regarding control) according to the semen origin and ASA titer in the blood sera of cows. Values were lower when thawed semen was used but no statistically ASA titer. Table 3. origin in cow s sera n = 11 It is also evident (Table 4) that total sperm motility was lower when samples of the cervical mucus with high ASA titer were analyzed despite the semen origin (native or thawed). Differences were higher when thawed semen was used for analysis. n = 15 n = 20 Blood sera Cervical mucus n = 10 Thawed semen n = 13 X± SD ± ± ± ±19.27 titer in cow s cervical mucus n = 22 X 69.01± ± ± ±24.72 Progressive sperm motility expressed the same pattern of changes as total motility. Motility was the smallest when thawed semen was incubated with cervical mucus samples containing high ASA titer (Table 5).

8 506 Acta Veterinaria (Beograd), Vol. 63, No. 5-6, , Table 5. origin in cows cervical mucus Like for the last two parameters, progressive sperm velocity (Table 6) was decreased when samples containing high ASA titers were analyzed with both native and thawed semen. As differences in the mean values for tail frequency movement and amplitude were not statistically different they are not presented here. The only exception was tail movement frequency when sera samples with low and high ASA titer were incubated with native or thawed semen (Table 8). Tables 7 and 8 contain results of the statistical analisis of differences between mean values of the estimated semen motility parameters. Cervical mucus n = 20 Thawed semen n = 22 X±SD 75.66± ± ± ±14.74 origin in cow s cervical mucus Cervical mucus n = 22 X± SD 86.71± ± ± ±18.64 Table 7. Statistical differences between sperm motility parameters regarding ASA titer values (low vs high titer) Cervical mucus Low vs high ASA titer Blood sera Low vs high ASA titer Total motility P < 0.05 NS Progressive motility P < 0.05 NS Progressive velocity NS NS Tail amplitude NS NS Frequency NS NS Thawed semen Total motility P < 0.05 NS Progressive motility P < 0.05 NS Progressive velocity P < 0.05 NS Tail amplitude NS NS Frequency NS NS

9 507 Total and progressive motility estimation of the native semen revealed that these paramers were lower following incubation with cervical mucus samples containing a high ASA titer. This was also evident when thawed samen was used and values were even lower. Moreover, with thawed semen, progressive sperm depending on the semen origin (native or thawed) following incubation with sera and cervical mucus samples with low and high ASA titer are presented in Table 8. Table 8. Statistical differences between sperm motility parameters regarding origin of the semen (native vs thawed semen) Cervical mucus Native vs thawed semen Blood sera Native vs thawed semen Total motility NS NS Progressive motility P < 0.01 P < 0.05 Progressive velocity NS P < 0.01 Tail amplitude NS NS Frequency NS P < 0.01 Total motility P < 0.05 NS Progressive motility P < 0.01 NS Progressive velocity NS NS Tail amplitude NS NS Frequency NS P < 0.01 Regarding ASA origin (cervical mucus or sera) progressive sperm motility for the test with both low and high ASA titer of cervical mucus samples. Values were much lower in samples with high ASA titer. In samples with high ASA titer high ASA titer progressive motility and progressive velocity were decreased when thawed semen was used for the test. Frequency of tail movements was also lower and interestingly this phenomenon was observed in sera samples with low ASA titer.

10 508 Acta Veterinaria (Beograd), Vol. 63, No. 5-6, , DISCUSSION (data not presented here). This emphases individual variability of the observed sperm motility characteristics and in some cases led to the lack of statistical at al. reported that the lenghth of open days period was et al., (2007) later on stated that the critical titre value of the antisperm Ig A antibodies from cervical mucus in cows is 1:64, as of AI in the last IgA ASA titer above this value. Rapid changes in intensive milk production during the last decades are dramatic and milk yield is constantly rising as a consequence of genetic selection, improved nutrition and management. However, it seems that cows with higher milk production have more reproductive problems. The most important causes lactation on reproduction and early embryonic death (Lucy, 2001). Recently, it milking cows, are closely related to negative energy balance (NEB) emerging from metabolic and hormonal changes due to extreme milk production. Therefore, a price for high productivity might be decreased fertility induced by hormonal might have a dominant role are LH, because its pulsations are decreased during Smith, 1989). Balendran et al. (2008) investigated progesterone concentrations fertility. According to Pryce et al. (2001), cows with lower BCS after calving are have reduced chances for conception and exactly in that period they are being inseminated, very often with no positive outcome. As it was proven in numerous studies, ASA titer increases with the age and total number of AI attempts, it is likely that cows might be isoimmunised with antigens originated from semen and/or extender partly because of seminal plasma lack. Immunological subfertility raised is our strong opinion that this hypothesis is realistic but so far, there are no reliable data to support it.

11 509 We are able to conclude that levels of the ASA are increasing with age and total number of AI attempts and that ASA in cervical mucus reduce sperm motility. Moreover, NEB reduces the chances for fertilization and a novel approach to this problem is needed to overcome the present situation. ACKNOWLEDGEMENT: This work was funded by Serbian Ministry for Science and Technological Development, Project No. III 46002, , Molecular genetic and ecophysiological researches on the protection of autochthonous animal resources, sustaining domestic animals welfare, health and reproduction, and safe food production Address for correspondence: Dr Miodrag Lazarevic, professor Faculty of Veterinary Medicine University of Belgrade Bul. oslobodjenja Belgrade, Serbia lazarevicm@vet.bg.ac.rs REFERENCES 1. Baledran A, Gordon M, Pretheeban T, Singh R, Perera R, Rajamahedran R, 2008, Decreased fertility with increasing parity in lactating dairy cows, J Can Anim Sci, 88, Bratanov K, Tornyov A, Hristova-Koleva M, 1975, Studies on the immunoglobulins in se cretion of the female reproductive tract, III Int Symp Immunol Reprod, Varna, Abstracts, Casida EL, 1961, Present status of the repeat breeder cow problem, J Dairy Sci, 44, , 1989, Interrelationships between energy balance and postpartum reproductive function in dairy cattle, J Dairy Sci, 72, Check JH, Adelson HG, Bollendorf A, 1991, Effect of antisperm antibodies on computerized semen analysis, Arch Androl, 27, Clarke GN antibodies blocking cervical mucus by human spermatozoa, Am J Reprod Immunol Microbiol, 16, Didion AB samples, Theriogenology, 70, , 1993, Antisperm antibodies in cervical mucus in an unselected subfertile population, Hum Reprod, 8, characteristics measured by computer-assisted sperm analysis (CASA) and relationship to fertility, Theriogenology, 49, Hogarth JP, 1982, Immunological aspects of mammalian reproduction, Blackie, Glasgow & London, , 2009, Effects of addition of vitamin B12 to the extender on post-thaw motility, acrosome morphology, and plasma membrane integrity in bull semen, Turk J Vet Anim Sci, 33, 5, indeksom osemenjavanja, Magistarski rad, Fakultet veterinarske medicine, Beograd. 13. Vojnosanit Pregl, 57, 3, , 1999, Effects of experimentally generated bull antisperm antibodies on in vitro fertilization, Biol Reprod, 60,

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13 511 UTICAJ ANTISPERMALNIH ANTITELA Ig G I Ig A KLASE POREKLOM IZ SERUMA I CERVIKALNE SLUZI KRAVA NA POKRETLJIVOST SPERMATOZOIDA protiv antigena spermatozoida (ASA) iz krvnog seruma i cervikalne sluzi krava na su bile rase buša ili melezi buše sa simentalcem. Titar antitela Ig G i Ig A klase krvnom serumu i plotkinje sa visokim i niskim titrom ASA u cervikalnoj sluzi. Uticaj (Computer Assisted Semen Analisis - CASA). spermatozoida u zavisnosti od njihovog porekla i titra.